Lysosome-related organelles.

Lysosomes are membrane-bound cytoplasmic organelles involved in intracellular protein degradation. They contain an assortment of soluble acid-dependent hydrolases and a set of highly glycosylated integral membrane proteins. Most of the properties of lysosomes are shared with a group of cell type-specific compartments referred to as 'lysosome-related organelles', which include melanosomes, lytic granules, MHC class II compartments, platelet-dense granules, basophil granules, azurophil granules, and Drosophila pigment granules. In addition to lysosomal proteins, these organelles contain cell type-specific components that are responsible for their specialized functions. Abnormalities in both lysosomes and lysosome-related organelles have been observed in human genetic diseases such as the Chediak-Higashi and Hermansky-Pudlak syndromes, further demonstrating the close relationship between these organelles. Identification of genes mutated in these human diseases, as well as in mouse and Drosophila: pigmentation mutants, is beginning to shed light on the molecular machinery involved in the biogenesis of lysosomes and lysosome-related organelles.     

Endobrevin/VAMP8 mediates exocytotic release of hexosaminidase from rat basophilic leukaemia cells

Mast cells are important players in innate immunity and mediate allergic responses. Upon stimulation, they release biologically active mediators including histamine, cytokines and lysosomal hydrolases. We used permeabilized rat basophilic leukaemia cells as model to identify R-SNAREs (soluble NSF (N-ethylmaleimide-sensitive fusion protein)) mediating exocytosis of hexosaminidase from mast cells. Of a complete set of recombinant mammalian R-SNAREs, only vesicle associated membrane protein (VAMP8)/endobrevin consistently blocked hexosaminidase release, which was also insensitive to treatment with clostridial neurotoxins. Thus, VAMP8, which also mediates fusion of late endosomes and lysosomes, plays a major role in hexosaminidase release, strengthening the view that mast cell granules share properties of both secretory granules and lysosomes.     

Vacuolization of mucolipidosis type II mouse exocrine gland cells represents accumulation of autolysosomes

We previously reported that mice deficient in UDP-GlcNAc:lysosomal enzyme GlcNAc-1-phosphotransferase (mucolipidosis type II or Gnptab -/- mice), the enzyme that initiates the addition of the mannose 6-phosphate lysosomal sorting signal on acid hydrolases, exhibited extensive vacuolization of their exocrine gland cells, while the liver, brain, and muscle appeared grossly unaffected. Similar pathological findings were observed in several exocrine glands of patients with mucolipidosis II. To understand the basis for this cell type-specific abnormality, we analyzed these tissues in Gnptab -/- mice using a combined immunoelectron microscopy and biochemical approach. We demonstrate that the vacuoles in the exocrine glands are enlarged autolysosomes containing undigested cytoplasmic material that accumulate secondary to deficient lysosomal function. Surprisingly, the acid hydrolase levels in these tissues ranged from normal to modestly decreased, in contrast to skin fibroblasts, which accumulate enlarged lysosomes and/or autolysosomes also but exhibit very low levels of acid hydrolases. We propose that the lysosomal defect in the exocrine cells is caused by the combination of increased secretion of the acid hydrolases via the constitutive pathway along with their entrapment in secretory granules. Taken together, our results provide new insights into the mechanisms of the tissue-specific abnormalities seen in mucolipidosis type II.     

Herpes simplex virus and human cytomegalovirus replication in WI-38 cells. III. Cytochemical localization of lysosomal enzymes in infected cells.

Cytochemical localization of the lysosomal enzymes acid phosphatase and arylsulfatase in cells infected by herpes simplex virus (HSV) or human cytomegalovirus (CMV) showed the following interactions between viruses and host cell lysosomes: (i) many enveloped progeny viruses were located within cytoplasmic vacuoles containing lysosomal enzyme activity; (ii) naked cytoplasmic capsids appeared to acquire an envelope by budding directly into lysosomes; and (iii) many of the cytoplasmic dense bodies that are characteristic of CMV-infected cells and are thought to represent noninfectious aggregates of CMV structural proteins (I. Sarov and I. Abady, Virology 66:464-473, 1975) also acquired a limiting membrane by budding into lysosomes. Autophagy of other cytoplasmic elements was not observed, suggesting that there is some specificity involved in the association of viral particles and CMV dense bodies with lysosomes. Despite the presence of potentially destructive hydrolases, there was little evidence of significant morphological damage to intralysosomal viruses, and high titers of infectious particles were released into the medium. It would therefore appear that significant levels of HSV and CMV infectivity normally persist even though many progeny particles are directly exposed to lysosomal enzymes.     

Autophagy regulates cholesterol efflux from macrophage foam cells via lysosomal acid lipase

The lipid droplet (LD) is the major site of cholesterol storage in macrophage foam cells and is a potential therapeutic target for the treatment of atherosclerosis. Cholesterol, stored as cholesteryl esters (CEs), is liberated from this organelle and delivered to cholesterol acceptors. The current paradigm attributes all cytoplasmic CE hydrolysis to the action of neutral CE hydrolases. Here, we demonstrate an important role for lysosomes in LD CE hydrolysis in cholesterol-loaded macrophages, in addition to that mediated by neutral hydrolases. Furthermore, we demonstrate that LDs are delivered to lysosomes via autophagy, where lysosomal acid lipase (LAL) acts to hydrolyze LD CE to generate free cholesterol mainly for ABCA1-dependent efflux; this process is specifically induced upon macrophage cholesterol loading. We conclude that, in macrophage foam cells, lysosomal hydrolysis contributes to the mobilization of LD-associated cholesterol for reverse cholesterol transport.     

Identification of a chymotrypsin-like proteinase in human mast cells

An antiserum was produced against a chymotryptic proteinase purified from human skin. The antiserum did not cross-react with human leukocyte cathepsin G and elastase, rat mast cell proteinase I, and human skin tryptase. Indirect immunofluorescent staining of frozen skin sections to localize the proteinase showed cytoplasmic staining of cells scattered about the papillary dermis and around blood vessels and appendages. Restaining these sections with toluidine blue revealed that the fluorescently stained cells contained metachromatically staining granules, the major distinguishing feature of mast cells. A similar correlation was found in lung tissue. Ultrastructural studies employing the ferritin bridge technique to immunologically identify the proteinase additionally localized the proteinase to mast cell granules. Biochemical and immunochemical characterization of chymotryptic activity solubilized from isolated human lung mast cells identified a chymotryptic proteinase that may be identical to the skin chymotryptic proteinase. These studies establish that human skin mast cells contain a chymotrypsin-like proteinase that is a granule constituent and provide evidence that indicates a comparable proteinase is also present in lung mast cells.     

High- and low-uptake forms of β-hexosaminidase in human platelets Selective retention of the high-uptake form during stimulation with thrombin

Human platelets are rich in β-hexosaminidase and other acid hydrolases contained in organelles (lysosomes) distinct from α-granules and dense granules. Incubation of platelets with bovine or human thrombin (100 U/ml for 5 min at 37°C) induces the secretion of 100% of the contents of α- and dense granules, but only 40–60% of total β-hexosaminidase from lysosomes. Both isozymes Hex A and Hex B are secreted in the same proportion as found intracellulary. There is no selective recapture or plasma membrane binding by platelets of secreted β-hexosaminidase. The secreted enzyme is of the low-uptake type, i.e., it is poorly recognized by the phosphomannosyl receptor-mediated uptake mechanism of fibroblasts, while the retained enzyme is a 3-fold higher uptake form. Preincubation of platelets with NH₄Cl (10 mM, 2 h), followed by thrombin stimulation, results in secretion of all β-hexosaminidase as a low-uptake form. The data support the hypothesis that there are secretory and nonsecretory forms of lysosomes. The secretory lysosomes would contain low-uptake forms of hydrolases in addition to acid phosphatase, while the nonsecretory lysosomes would contain high-uptake hydrolases and be acid phosphatase-deficient. Conditions where the contents of both lysosomal populations were released together, i.e., amine treatment followed by thrombin induction, or extraction of unstimulated cells, would result in the exposure of high-uptake phosphomannosylated hydrolases released from one population of lysosomes to acid phosphatase released from the second population of lysosomes with their subsequent conversion to low-uptake forms.     

The sorting and trafficking of lysosomal proteins

For a long time lysosomes were considered terminal organelles involved in the degradation of different substrates. However, this view is rapidly changing by evidence demonstrating that these organelles and their content display specialized functions in addition to the degradation of substances. Many lysosomal proteins have been implicated in specialized cellular functions and disorders such as antigen processing, targeting of surfactant proteins, and most lysosomal storage disorders. To date, about fifty lysosomal hydrolases have been identified, and the majority of them are targeted to the lysosomes via the mannose-6-phosphate receptor (M6P-Rc). However, recent studies on the intracellular trafficking of the non-enzymic lysosomal proteins prosaposin and GM2 activator (GM2AP) demonstrated that they use an alternative receptor termed "sortilin". Existing evidence suggests that some hydrolases traffic to the lysosomes in a mannose 6-phophate-indepentend manner. The possibility that sortilin is implicated in the targeting of some soluble hydrolases, as well as the consequences of this process, is addressed in the present review.     

The effects of sucrose loading on lysosomal hydrolases

Summary The addition of 88 mM sucrose to the culture medium of human skin fibroblasts from normal subjects caused remarkable increase in the intracellular lysosomal hydrolase activities. The mechanism of this induction by sucrose loading was carefully studied with several fibroblast strains of different inherited lysosomal storage disorders. In single lysosomal hydrolase defect such as GM₁-gangliosidosis, mannosidosis and Sandhoff disease, no induction of the deficient hydrolase was found with 88 mM sucrose loading. In contrast, sucrose loading caused normalization of intracellular lysosomal hydrolase activities in I-cell disease fibroblasts and cytoplasmic inclusion materials disappeared. Subsequent investigations reveal that I-cell disease cells are classified into three subgroups by the degree of hydrolase induction by sucrose loading; a high responding, an intermediate responding and a no-response group. The heterogeneity may be based upon different induction by sucrose loading of the enzyme, probably the residual phosphotransferase which is involved in the processing steps of lysosomal enzyme molecules. With the addition of mannose-6-phosphate and 10 mM NH₄Cl to cultured skin fibroblasts, it was shown that sucrose loading caused increased synthesis of lysosomal enzyme proteins. The result of the test with 2,4-dinitrophenol suggests that sucrose is indeed pinocytosed by cultured human skin fibroblasts and localized in lysosomes and that this event is the essential factor to trigger the induction of lysosomal hydrolases. Simultaneous loading of both invertase and sucrose in cultured cells caused no induction of -mannosidase activity. This result indicates that invertase is also pinocytosed, reaches the lysosomes and hydrolyzes sucrose in the lysosomes. Lysosomal overloading with sucrose resulted in induction of lysosomal hydrolases and invertase blocked the induction of -mannosidase activity. However, some induction still exists in -galactosidase and -fucosidase activity. Thus it is very likely that the induction of lysosomal hydrolases demands a complicated process.In this article, we investigated the effects of sucrose on the lysosomal hydrolases in cultured human skin fibroblasts of several inherited lysosomal storage disorders and normal subjects and discuss the possible mechanism. of the induction of lysosomal hydrolase activities by sucrose loading.     

Modulation of LXR-α and the effector genes by Ascorbic acid and Statins in psoriatic keratinocytes

Following the degradative pathway, vesicles loaded with extracellular material, eventually, dock and fuse with lysosomes, acquiring specific membrane markers of these organelles and acid hydrolases responsible for digest their content. The lysosomal-associated membrane protein 2 (LAMP-2), the best characterized lysosomal membrane protein, is found in late stages of endosome maturation and may be used as a marker of lysosome-associated membranes. Lysosomal storage disorders (LSDs) are described by the absence or deficiency in hydrolase activity leading to substrate accumulation within lysosomal components and to the onset of several diseases. It is known that lymphocytes infected by Epstein–Barr virus (EBV) are able to form cytoplasmic vacuoles, which work as a storage compartment for lysosomal acidic hydrolases. At the present study, we validate the EBV as a transforming agent of B lymphocytes in stability studies of long-term stored samples, since the methods used to keep samples in liquid nitrogen and thaw them have all proven to be efficient in samples frozen for up to 2 years. To confirm and investigate some of the most prevalent LSDs in the South of Brazil—Pompe, Fabry and Gaucher diseases—we first measured the enzymatic activity of α-glicosidase, α-galactosidase, and β-glicosidase in those cytoplasmic-formed vacuoles and then looked to LAMP-2 immunoreactivity by employing confocal microscopy techniques.     

Retrieval of lysosomal membrane and acid phosphatase from phagolysosomes of Paramecium caudatum

Little is known about the fate of lysosomal membrane in phagocytic cells. Because the age of the digestive vacuoles in Paramecium caudatum can be easily determined, we have been able to study the dynamic membrane events in the older vacuoles. Late in the phagolysosomal stage (DV-III) the vacuole membrane undergoes a burst of tubule formation. The tubules expand into vesicles which have characteristics resembling lysosomes in both thin sections and freeze-fracture replicas. The tubules also contain acid phosphatase activity when they arise from acid phosphatase-reactive vacuoles. We conclude that after active digestion lysosomal membrane is retrieved in whole or in part along with some membrane-associated hydrolases. A logical extension of these results is that the lysosome-like vesicles, after being recharged with hydrolases by fusing with primary lysosomes, are recycled back to DV-II for reuse.     

A mouse model for mucopolysaccharidosis type III A (Sanfilippo syndrome)

Mucopolysaccharidosis type III A (MPS III A, Sanfilippo syndrome) is a rare, autosomal recessive, lysosomal storage disease characterized by accumulation of heparan sulfate secondary to defective function of the lysosomal enzyme heparan N- sulfatase (sulfamidase). Here we describe a spontaneous mouse mutant that replicates many of the features found in MPS III A in children. Brain sections revealed neurons with distended lysosomes filled with membranous and floccular materials with some having a classical zebra body morphology. Storage materials were also present in lysosomes of cells of many other tissues, and these often stained positively with periodic-acid Schiff reagent. Affected mice usually died at 7-10 months of age exhibiting a distended bladder and hepatosplenomegaly. Heparan sulfate isolated from urine and brain had nonreducing end glucosamine- N -sulfate residues that were digested with recombinant human sulfamidase. Enzyme assays of liver and brain extracts revealed a dramatic reduction in sulfamidase activity. Other lysosomal hydrolases that degrade heparan sulfate or other glycans and glycosaminoglycans were either normal, or were somewhat increased in specific activity. The MPS III A mouse provides an excellent model for evaluating pathogenic mechanisms of disease and for testing treatment strategies, including enzyme or cell replacement and gene therapy.     

Lysosomal integral membrane glycoproteins are expressed at high levels in the inclusion bodies of I-cell disease fibroblasts

The localization, expression, and transport of two lysosomal integral membrane glycoproteins of human cells, hLAMP-1 and hLAMP-2, have been studied in mucolipidosis II (I-cell disease) fibroblasts. These cells are deficient in N-acetylglucosaminylphosphotransferase, one of the enzymes required for addition of the mannose 6-phosphate recognition signal to newly synthesized lysosomal hydrolases and a prerequisite for the sorting and transport of the hydrolases to lysosomes. I-cells analyzed by immunofluorescence microscopy with monoclonal antibodies against hLAMP-1 and hLAMP-2 showed intense staining of the inclusion bodies covering most of the cytoplasm of the cells. Immunoelectron microscopy confirmed this localization and showed that the hLAMP-positive vesicles commonly contained membrane structures or electron-dense homogeneous material characteristic of secondary lysosomes. Studies of the biosynthesis of hLAMP-2 in I-cells pulse-labeled with [35S]methionine indicated that the molecule is glycosylated in the Golgi system, is transported to vesicles with the high density characteristic of lysosomes, and has chemical properties similar to those of the glycoprotein synthesized in normal cells. The concentration of the hLAMP-2 glycoprotein was three- to fourfold greater than that in normal fibroblasts, in sharp contrast to the reduced levels of lysosomal hydrolases seen in I-cells. These experiments demonstrate that the inclusion bodies in I-cells have properties of secondary lysosomes and that the transport and targeting of the lysosomal membrane glycoproteins to the inclusion bodies of these cells is not coupled to the mannose 6-phosphate system for transporting soluble acid hydrolases.     

Ultrastructural cytochemistry of chronic T-cell leukaemias. A study with four acid hydrolases

The ultrastructural localization of four acid hydrolases (acid phosphatase, beta-glucuronidase, beta-glucosaminidase and alpha-naphthylacetate esterase) has been studied in lymphocytes from 16 patients with three types of chronic T-cell leukaemia, namely, T-prolymphocytic leukaemia (T-PLL), T-chronic lymphocytic leukaemia (T-CLL) and adult T-cell lymphoma leukaemia (ATLL). Different patterns of enzyme distribution were observed in the leukaemic T-cells from these disorders. In T-PLL, reactivity for the four acid hydrolases was confined to single or a few large granules. Gall bodies were reactive for beta-glucuronidase, b-glucosaminidase and alpha-naphthylacetate esterase but apparently unreactive for acid phosphatase. In T-CLL, scattered small- to medium-size cytoplasmic granules and many parallel tubular arrays were strongly reactive for acid phosphatase, beta-glucuronidase and beta-glucosidase but showed no reactivity for alpha-naphthylacetate esterase. Intermediate features were observed in ATLL. The observed differences in enzyme reactivity reflect a different content of lysosomal granules in the various types of leukaemic T-cells. They also suggest that similar differences may be found in normal T-lymphocyte subsets.     

Lysosomes in iron metabolism,ageing and apoptosis

The lysosomal compartment is essential for a variety of cellular functions, including the normal turnover of most long-lived proteins and all organelles. The compartment consists of numerous acidic vesicles (pH ∼4 to 5) that constantly fuse and divide. It receives a large number of hydrolases (∼50) from the trans-Golgi network, and substrates from both the cells’ outside (heterophagy) and inside (autophagy). Many macromolecules contain iron that gives rise to an iron-rich environment in lysosomes that recently have degraded such macromolecules. Iron-rich lysosomes are sensitive to oxidative stress, while ‘resting’ lysosomes, which have not recently participated in autophagic events, are not. The magnitude of oxidative stress determines the degree of lysosomal destabilization and, consequently, whether arrested growth, reparative autophagy, apoptosis, or necrosis will follow. Heterophagy is the first step in the process by which immunocompetent cells modify antigens and produce antibodies, while exocytosis of lysosomal enzymes may promote tumor invasion, angiogenesis, and metastasis. Apart from being an essential turnover process, autophagy is also a mechanism by which cells will be able to sustain temporary starvation and rid themselves of intracellular organisms that have invaded, although some pathogens have evolved mechanisms to prevent their destruction. Mutated lysosomal enzymes are the underlying cause of a number of lysosomal storage diseases involving the accumulation of materials that would be the substrate for the corresponding hydrolases, were they not defective. The normal, low-level diffusion of hydrogen peroxide into iron-rich lysosomes causes the slow formation of lipofuscin in long-lived postmitotic cells, where it occupies a substantial part of the lysosomal compartment at the end of the life span. This seems to result in the diversion of newly produced lysosomal enzymes away from autophagosomes, leading to the accumulation of malfunctioning mitochondria and proteins with consequent cellular dysfunction. If autophagy were a perfect turnover process, postmitotic ageing and several age-related neurodegenerative diseases would, perhaps, not take place.     

Localization of nitric oxide synthase immunoreactivity in mast cells of human nasal mucosa

Nitric oxide (NO)-synthase immunoreactivity has been detected for the first time in mast cells of human normal nasal mucosa, with an antibody specific for neuronal NO-synthase. Intense immunoreactivity was revealed in secretion granules of mast cells but was found in mast cell granules free in the extracellular matrix only in some instances; no reactivity was found in the cytoplasm of this or other cell types. These findings suggest that human nasal mast cells contain a particulate isoform of NO-synthase, which shares epitopes with neuronal NO-synthase and is rapidly removed from granules upon exocytosis.     

Lysosomal pathways to cell death and their therapeutic applications

Lysosomes are the major cell digestive organelles that were discovered over 50 years ago. They contain a number of hydrolases that help them to degrade intracellular and extracellular material delivered. Among the hydrolases, the cathepsins, a group of proteases enclosed in the lysosomes, have a major role. About a decade ago, the cathepsins were found to participate in apoptosis. Following their release into the cytosol, they cleave Bid and degrade antiapoptotic Bcl-2 proteins, thereby triggering the mitochondrial pathway of apoptosis, with the lysosomal membrane permeabilization being the critical step in this pathway. Lysosomal dysfunction is linked with several diseases, including cancer and neurodegenerative disorders, thereby providing a potential for therapeutic applications. In this review lysosomes and lysosomal proteases involvement in apoptosis and their possible pharmaceutical targeting are discussed.     

Distinct molecular mechanisms for protein sorting within immature secretory granules of pancreatic beta-cells

In the beta-cells of pancreatic islets, insulin is stored as the predominant protein within storage granules that undergo regulated exocytosis in response to glucose. By pulse-chase analysis of radiolabeled protein condensation in beta-cells, the formation of insoluble aggregates of regulated secretory protein lags behind the conversion of proinsulin to insulin. Condensation occurs within immature granules (IGs), accounting for passive protein sorting as demonstrated by constitutive-like secretion of newly synthesized C- peptide in stoichiometric excess of insulin (Kuliawat, R., and P. Arvan. J. Cell Biol. 1992. 118:521-529). Experimental manipulation of condensation conditions in vivo reveals a direct relationship between sorting of regulated secretory protein and polymer assembly within IGs. By contrast, entry from the trans-Golgi network into IGs does not appear especially selective for regulated secretory proteins. Specifically, in normal islets, lysosomal enzyme precursors enter the stimulus-dependent secretory pathway with comparable efficiency to that of proinsulin. However, within 2 h after synthesis (the same period during which proinsulin processing occurs), newly synthesized hydrolases are fairly efficiently relocated out of the stimulus- dependent pathway. In tunicamycin-treated islets, while entry of new lysosomal enzymes into the regulated secretory pathway continues unperturbed, exit of nonglycosylated hydrolases from this pathway does not occur. Consequently, the ultimate targeting of nonglycosylated hydrolases in beta-cells is to storage granules rather than lysosomes. These results implicate a post-Golgi mechanism for the active removal of lysosomal hydrolases away from condensed granule contents during the storage process for regulated secretory proteins.     

A fascination with sugars

We now recognize that a large number of membrane and soluble proteins contain covalently linked oligosaccharides that exhibit a vast array of structures and participate in a wide variety of biological processes. Nowhere is this better illustrated than the mannose 6-phosphate (Man-6-P) recognition system that mediates the trafficking of newly synthesized acid hydrolases to lysosomes in higher eukaryotes. The Asn-linked high-mannose oligosaccharides of these hydrolases facilitate folding of the nascent proteins in the endoplasmic reticulum via interaction with lectin-type chaperones and after phosphorylation in the Golgi, function as ligands for binding to Man-6-P receptors, a critical step in their transport to lysosomes. Failure to synthesize the Man-6-P recognition marker results in a serious lysosomal storage disease, one of a growing number of genetic conditions, termed congenital disorders of glycosylation, that result from faulty glycan biosynthesis.     

Cell- and ligand-specific dephosphorylation of acid hydrolases: evidence that the mannose 6-phosphatase is controlled by compartmentalization

Mouse L cells that possess the cation-independent mannose 6-phosphate (Man 6-P)/insulin-like growth factor (IGF) II receptor change the extent to which they dephosphorylate endocytosed acid hydrolases in response to serum (Einstein, R., and C. A. Gabel. 1989. J. Cell Biol. 109:1037-1046). To investigate the mechanism by which dephosphorylation competence is regulated, the dephosphorylation of individual acid hydrolases was studied in Man 6-P/IGF II receptor-positive and -deficient cell lines. 125I-labeled Man 6-P-containing acid hydrolases were proteolytically processed but remained phosphorylated when endocytosed by receptor-positive L cells maintained in the absence of serum; after the addition of serum, however, the cell-associated hydrolases were dephosphorylated. Individual hydrolases were dephosphorylated at distinct rates and to different extents. In contrast, the same hydrolases were dephosphorylated equally and completely after entry into Man 6-P/IGF II receptor-positive Chinese hamster ovary (CHO) cells. The dephosphorylation competence of Man 6-P/IGF II receptor-deficient mouse J774 cells was more limited. beta-Glucuronidase produced by these cells underwent a limited dephosphorylation in transit to lysosomes such that diphosphorylated oligosaccharides were converted to monophosphorylated species. The overall quantity of phosphorylated oligosaccharides associated with the enzyme, however, did not decrease within the lysosomal compartment. Likewise, beta-glucuronidase was not dephosphorylated when introduced into J774 cells via Fc receptor-mediated endocytosis. The CHO and J774 cell lysosomes, therefore, display opposite extremes with respect to their capacity to dephosphorylate acid hydrolases; within CHO cell lysosomes acid hydrolases are rapidly and efficiently dephosphorylated, but within J774 cell lysosomes the same acid hydrolases remain phosphorylated. This difference in processing indicates that lysosomes themselves exist in a dephosphorylation-competent and -incompetent state. Man 6-P-bearing acid hydrolases endocytosed by the L+ cells in the absence of serum were not distributed uniformly throughout the lysosomal compartment. The change in the dephosphorylation competence of L cells in response to serum suggests, therefore, that these cells contain multiple populations of lysosomes that differ with respect to their content of a mannose 6-phosphatase, and that serum factors affect the distribution of hydrolases between the different compartments.